This invention relates to a novel human macrophage metalloproteinase having elastolytic activity and, more particularly, to the cDNA clone representing the 54 kDa proenzyme referred to herein as human macrophage metalloelastase (HME).
Metalloproteinases comprise a family of structurally related matrix degrading enzymes involved in normal embryonic development, growth, tissue remodeling, and tissue repair.
Macrophages participate in extracellular matrix turnover both directly by secretion of proteinases and proteinase inhibitors and indirectly by the elaboration of cytokines such as interleukin-1 or tumor necrosis factor, which induces proteinase production by resident fibroblasts. Human macrophages have the capacity to produce several metalloproteinases including interstitial collagenase, stromelysin, and two type IV collagenase/gelatinases of 92 and 72 kDa, of which 92 kDa enzyme represents the predominant macrophage product. The DNA cloning of interstitial collagenase and the two type IV-collagenase/gelatinases of 72 kDa and 92 kDa is described, e.g., in U.S. Pat. Nos. 4,772,557, 4,923,818 and 4,992,537, respectively. The DNA cloning of stromelysin is described in PCT Int. Appln. WO 87 07,907, published Dec. 30, 1987.
Macrophages also produce the counter-regulatory tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), which are proteins that inhibit metalloproteinases via a high affinity, noncovalent bond. TIMP-2 and its homology to TIMP-1 are described in EP 404,750, published Dec. 27, 1990 and in copending U.S. application Ser. No. 07/358,043, filed May 26, 1989.
Elastin degradation and abnormal repair are pivotal events in the pathogenesis of pulmonary emphysema. Cigarette smoking, the predominant risk factor for emphysema, is associated with a large accumulation of macrophages in the lungs. Cigarette smoking is associated with a 10-fold increase in cells recovered by bronchoalveolar lavage [Hunninghake and Crystal, Am. Rev. Resp. Dis. 128, 833-838 (1990)] with macrophages comprising over 98% of cells and PMNs less than 1% [Merchant et al., Ibid. 146, 448-453 (1992)]. Moreover, macrophages are predominant in the respiratory bronchioles of cigarette smokers where emphysematous changes are first manifest [Niewoehner et al., N. Engl. J. Med. 291, 755-758 (1974)].
Recently, two classes of macrophage-derived proteinases have been implicated in elastolysis: (1) Matrix metalloproteinases (MMPs) that can degrade elastin include the 92 kDa gelatinase, an abundant macrophage product, and the 72 kDa gelatinase, secreted by many cell types but present in only trace amounts in macrophages [Welgus et al., J. Clin. Invest. 86, 1496-1502 (1990); Murphy and Docherty, Am. J. Respir. Cell Mol. Biol. 7, 120-125 (1992); and Senior et al., J. Biol. Chem. 266, 7870-7875 (1991)]. Matrilysin, another recently described metalloproteinase with elastolytic activity, is found in peripheral blood monocytes but not in alveolar macrophages [Busiek et al., J. Biol. Chem. 267, 9087-9092 (1992)]. (2) Cysteine proteinases produced by macrophages that cleave elastin include cathepsin L [Reilly et al., Biochem. J. 257, 493-498 (1989)] and cathepsin S [Shi et al., J. Biol. Chem. 267, 7258-7262 (1992)], both of which are most active in acidic environments (.about.pH 5).
It has been reported that media conditioned by mouse peritoneal macrophages exhibited significant elastolytic activity [Werb and Gordon, J. Exp. Med. 142, 361-377 (1975)]. A 22 kDa, metal-dependent, elastolytic proteinase, termed mouse macrophage elastase (MME), was isolated [Banda and Werb, Biochem. J. 193, 589-605 (1981)]. However, despite the efforts of many investigators, human macrophage elastase activity could not be documented and many doubted its existence. The present inventor and colleagues recently cloned the MME cDNA and demonstrated that MME is truly a member of the MMP family [Shapiro et al., J. Biol. Chem. 267, 4664-4671 (1992)]. Surprisingly, the molecular mass of the MME proenzyme is 53 kDa, similar to several other MMPs including the collagenases and stromelysins. The 22 kDa active form of MME, previously described, results from both classic N-terminal activation and unusual C-terminal processing. MME is less than 50% identical at the amino acid level to all known human MMPs.